qqwang-berkeley / JUM

A tool for annotation-free differential analysis of tissue-specific pre-mRNA alternative splicing patterns

MIT License

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Error when running JUM step A #52

Open hvicars opened 5 months ago

hvicars commented 5 months ago

Hi,

I keep encountering an error while running JUM step A. I am using JUM 3.0.0. The error is: "ERROR: Sort order was unspecified, and file treat1Aligned.out.sorted.bed is not sorted lexicographically. Please rerun with the -g option for a genome file. See documentation for details."

The error appears after line 238. I did STAR mapping for my control and treatment RNA seq data sets before running JUM step A.

Please let me know if there is any other information you need from me or if you have any idea how I might be able to fix this error.

Thank you so much!

qqwang-berkeley commented 5 months ago

Hi,

Did you sort the bam files after STAR mapping?

Convert and sort the resulted alignment files (please use the exact naming nomenclature as shown below). Here we show ctrl 1 sample as an example but users need to do this for all the samples:

$ samtools view -bS ctrl1Aligned.out.sam > ctrl1Aligned.out.bam $ samtools sort -o ctrl1Aligned.out_sorted.bam -T ctrl1_temp ctrl1Aligned.out.bam $ samtools index ctrl1Aligned.out_sorted.bam $ rm *Aligned.out.bam

Qingqing

On Wed, Feb 28, 2024 at 5:55 PM hvicars @.***> wrote:

Hi,

I keep encountering an error while running JUM step A. I am using JUM 3.0.0. The error is: "ERROR: Sort order was unspecified, and file treat1Aligned.out.sorted.bed is not sorted lexicographically. Please rerun with the -g option for a genome file. See documentation for details."

The error appears after line 238. I did STAR mapping for my control and treatment RNA seq data sets before running JUM step A.

Please let me know if there is any other information you need from me or if you have any idea how I might be able to fix this error.

Thank you so much!

— Reply to this email directly, view it on GitHub https://github.com/qqwang-berkeley/JUM/issues/52, or unsubscribe https://github.com/notifications/unsubscribe-auth/AGJ6PW6HL3YQOY2GLPUGXQDYV6YVHAVCNFSM6AAAAABD64RFQOVHI2DSMVQWIX3LMV43ASLTON2WKOZSGE3DAMBRGQ4TGNA . You are receiving this because you are subscribed to this thread.Message ID: @.***>

hvicars commented 5 months ago

Hi Qingqing,

Yes, I used the script below to convert and sort the alignment files.

Adding module

module add samtools/1.17

samtools view -bS noHSAligned.out.sam > noHSAligned.out.bam samtools sort -o noHSAligned.out_sorted.bam -T noHS_temp noHSAligned.out.bam samtools index noHSAligned.out_sorted.bam

samtools view -bS 16hrAligned.out.sam > 16hrAligned.out.bam samtools sort -o 16hrAligned.out_sorted.bam -T 16hr_temp 16hrAligned.out.bam samtools index 16hrAligned.out_sorted.bam

rm *Aligned.out.bam

echo "All files are now converted and sorted !"

Is there anything I should add to this script to ensure the files get sorted correctly?

Thank you! Hannah

-- Hannah Vicars, PhD (she/her) Postdoctoral Scholar, Fuller Lab Stanford University

From: Qingqing Wang @.> Date: Wednesday, February 28, 2024 at 8:10 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue #52) Hi,

Did you sort the bam files after STAR mapping?

Convert and sort the resulted alignment files (please use the exact naming nomenclature as shown below). Here we show ctrl 1 sample as an example but users need to do this for all the samples:

Qingqing

On Wed, Feb 28, 2024 at 5:55 PM hvicars @.***> wrote:

Hi,

I keep encountering an error while running JUM step A. I am using JUM 3.0.0. The error is: "ERROR: Sort order was unspecified, and file treat1Aligned.out.sorted.bed is not sorted lexicographically. Please rerun with the -g option for a genome file. See documentation for details."

The error appears after line 238. I did STAR mapping for my control and treatment RNA seq data sets before running JUM step A.

Please let me know if there is any other information you need from me or if you have any idea how I might be able to fix this error.

Thank you so much!

— Reply to this email directly, view it on GitHub https://github.com/qqwang-berkeley/JUM/issues/52, or unsubscribe https://github.com/notifications/unsubscribe-auth/AGJ6PW6HL3YQOY2GLPUGXQDYV6YVHAVCNFSM6AAAAABD64RFQOVHI2DSMVQWIX3LMV43ASLTON2WKOZSGE3DAMBRGQ4TGNA . You are receiving this because you are subscribed to this thread.Message ID: @.***>

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qqwang-berkeley commented 5 months ago

OK. What is the sequencing length of your read fastq file? Are all the sequencing reads of equal length?

On Fri, Mar 1, 2024 at 2:23 PM hvicars @.***> wrote:

Hi Qingqing,

Yes, I used the script below to convert and sort the alignment files.

Adding module

module add samtools/1.17

samtools view -bS noHSAligned.out.sam > noHSAligned.out.bam samtools sort -o noHSAligned.out_sorted.bam -T noHS_temp noHSAligned.out.bam samtools index noHSAligned.out_sorted.bam

samtools view -bS 16hrAligned.out.sam > 16hrAligned.out.bam samtools sort -o 16hrAligned.out_sorted.bam -T 16hr_temp 16hrAligned.out.bam samtools index 16hrAligned.out_sorted.bam

rm *Aligned.out.bam

echo "All files are now converted and sorted !"

Is there anything I should add to this script to ensure the files get sorted correctly?

Thank you! Hannah

-- Hannah Vicars, PhD (she/her) Postdoctoral Scholar, Fuller Lab Stanford University

From: Qingqing Wang @.> Date: Wednesday, February 28, 2024 at 8:10 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

Hi,

Did you sort the bam files after STAR mapping?

Convert and sort the resulted alignment files (please use the exact naming nomenclature as shown below). Here we show ctrl 1 sample as an example but users need to do this for all the samples:

$ samtools view -bS ctrl1Aligned.out.sam > ctrl1Aligned.out.bam $ samtools sort -o ctrl1Aligned.out_sorted.bam -T ctrl1_temp ctrl1Aligned.out.bam $ samtools index ctrl1Aligned.out_sorted.bam $ rm *Aligned.out.bam

Qingqing

On Wed, Feb 28, 2024 at 5:55 PM hvicars @.***> wrote:

Hi,

I keep encountering an error while running JUM step A. I am using JUM 3.0.0. The error is: "ERROR: Sort order was unspecified, and file treat1Aligned.out.sorted.bed is not sorted lexicographically. Please rerun with the -g option for a genome file. See documentation for details."

The error appears after line 238. I did STAR mapping for my control and treatment RNA seq data sets before running JUM step A.

Please let me know if there is any other information you need from me or if you have any idea how I might be able to fix this error.

Thank you so much!

— Reply to this email directly, view it on GitHub https://github.com/qqwang-berkeley/JUM/issues/52, or unsubscribe < https://github.com/notifications/unsubscribe-auth/AGJ6PW6HL3YQOY2GLPUGXQDYV6YVHAVCNFSM6AAAAABD64RFQOVHI2DSMVQWIX3LMV43ASLTON2WKOZSGE3DAMBRGQ4TGNA>

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hvicars commented 5 months ago

The sequencing length of my read fastq files is 101 bases. Yes, all reads are of equal length.

Hannah Vicars, PhD (she/her) Postdoctoral Scholar, Fuller Lab Stanford University

From: Qingqing Wang @.> Sent: Sunday, March 3, 2024 8:06:56 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>; Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue #52)

OK. What is the sequencing length of your read fastq file? Are all the sequencing reads of equal length?

On Fri, Mar 1, 2024 at 2:23 PM hvicars @.***> wrote:

Hi Qingqing,

Yes, I used the script below to convert and sort the alignment files.

Adding module

module add samtools/1.17

samtools view -bS noHSAligned.out.sam > noHSAligned.out.bam samtools sort -o noHSAligned.out_sorted.bam -T noHS_temp noHSAligned.out.bam samtools index noHSAligned.out_sorted.bam

samtools view -bS 16hrAligned.out.sam > 16hrAligned.out.bam samtools sort -o 16hrAligned.out_sorted.bam -T 16hr_temp 16hrAligned.out.bam samtools index 16hrAligned.out_sorted.bam

rm *Aligned.out.bam

echo "All files are now converted and sorted !"

Is there anything I should add to this script to ensure the files get sorted correctly?

Thank you! Hannah

-- Hannah Vicars, PhD (she/her) Postdoctoral Scholar, Fuller Lab Stanford University

From: Qingqing Wang @.> Date: Wednesday, February 28, 2024 at 8:10 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

Hi,

Did you sort the bam files after STAR mapping?

Convert and sort the resulted alignment files (please use the exact naming nomenclature as shown below). Here we show ctrl 1 sample as an example but users need to do this for all the samples:

$ samtools view -bS ctrl1Aligned.out.sam > ctrl1Aligned.out.bam $ samtools sort -o ctrl1Aligned.out_sorted.bam -T ctrl1_temp ctrl1Aligned.out.bam $ samtools index ctrl1Aligned.out_sorted.bam $ rm *Aligned.out.bam

Qingqing

On Wed, Feb 28, 2024 at 5:55 PM hvicars @.***> wrote:

Hi,

I keep encountering an error while running JUM step A. I am using JUM 3.0.0. The error is: "ERROR: Sort order was unspecified, and file treat1Aligned.out.sorted.bed is not sorted lexicographically. Please rerun with the -g option for a genome file. See documentation for details."

The error appears after line 238. I did STAR mapping for my control and treatment RNA seq data sets before running JUM step A.

Please let me know if there is any other information you need from me or if you have any idea how I might be able to fix this error.

Thank you so much!

— Reply to this email directly, view it on GitHub https://github.com/qqwang-berkeley/JUM/issues/52, or unsubscribe < https://github.com/notifications/unsubscribe-auth/AGJ6PW6HL3YQOY2GLPUGXQDYV6YVHAVCNFSM6AAAAABD64RFQOVHI2DSMVQWIX3LMV43ASLTON2WKOZSGE3DAMBRGQ4TGNA>

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qqwang-berkeley commented 5 months ago

That is really bizarre - it should work then if you aligned the reads against the same indexed genome and sorted them properly.

Which version of bedtools and samtools did you use?

On Mon, Mar 4, 2024 at 4:29 PM hvicars @.***> wrote:

The sequencing length of my read fastq files is 101 bases. Yes, all reads are of equal length.

Hannah Vicars, PhD (she/her) Postdoctoral Scholar, Fuller Lab Stanford University

From: Qingqing Wang @.> Sent: Sunday, March 3, 2024 8:06:56 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>; Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

OK. What is the sequencing length of your read fastq file? Are all the sequencing reads of equal length?

On Fri, Mar 1, 2024 at 2:23 PM hvicars @.***> wrote:

Hi Qingqing,

Yes, I used the script below to convert and sort the alignment files.

Adding module

module add samtools/1.17

samtools view -bS noHSAligned.out.sam > noHSAligned.out.bam samtools sort -o noHSAligned.out_sorted.bam -T noHS_temp noHSAligned.out.bam samtools index noHSAligned.out_sorted.bam

samtools view -bS 16hrAligned.out.sam > 16hrAligned.out.bam samtools sort -o 16hrAligned.out_sorted.bam -T 16hr_temp 16hrAligned.out.bam samtools index 16hrAligned.out_sorted.bam

rm *Aligned.out.bam

echo "All files are now converted and sorted !"

Is there anything I should add to this script to ensure the files get sorted correctly?

Thank you! Hannah

-- Hannah Vicars, PhD (she/her) Postdoctoral Scholar, Fuller Lab Stanford University

From: Qingqing Wang @.> Date: Wednesday, February 28, 2024 at 8:10 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

Hi,

Did you sort the bam files after STAR mapping?

Convert and sort the resulted alignment files (please use the exact naming nomenclature as shown below). Here we show ctrl 1 sample as an example but users need to do this for all the samples:

$ samtools view -bS ctrl1Aligned.out.sam > ctrl1Aligned.out.bam $ samtools sort -o ctrl1Aligned.out_sorted.bam -T ctrl1_temp ctrl1Aligned.out.bam $ samtools index ctrl1Aligned.out_sorted.bam $ rm *Aligned.out.bam

Qingqing

On Wed, Feb 28, 2024 at 5:55 PM hvicars @.***> wrote:

Hi,

I keep encountering an error while running JUM step A. I am using JUM 3.0.0. The error is: "ERROR: Sort order was unspecified, and file treat1Aligned.out.sorted.bed is not sorted lexicographically. Please rerun with the -g option for a genome file. See documentation for details."

The error appears after line 238. I did STAR mapping for my control and treatment RNA seq data sets before running JUM step A.

Please let me know if there is any other information you need from me or if you have any idea how I might be able to fix this error.

Thank you so much!

— Reply to this email directly, view it on GitHub https://github.com/qqwang-berkeley/JUM/issues/52, or unsubscribe <

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hvicars commented 5 months ago

For STAR mapping, I used:

STAR/2.7.10b samtools/1.17

For JUM step A, I used:

perl/5.34.1 samtools/1.19 bedtools/2.27.1 r/4.0 star/2.7.10b

From: Qingqing Wang @.> Date: Tuesday, March 5, 2024 at 6:23 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue #52) That is really bizarre - it should work then if you aligned the reads against the same indexed genome and sorted them properly.

Which version of bedtools and samtools did you use?

On Mon, Mar 4, 2024 at 4:29 PM hvicars @.***> wrote:

The sequencing length of my read fastq files is 101 bases. Yes, all reads are of equal length.

Hannah Vicars, PhD (she/her) Postdoctoral Scholar, Fuller Lab Stanford University

From: Qingqing Wang @.> Sent: Sunday, March 3, 2024 8:06:56 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>; Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

OK. What is the sequencing length of your read fastq file? Are all the sequencing reads of equal length?

On Fri, Mar 1, 2024 at 2:23 PM hvicars @.***> wrote:

Hi Qingqing,

Yes, I used the script below to convert and sort the alignment files.

Adding module

module add samtools/1.17

samtools view -bS noHSAligned.out.sam > noHSAligned.out.bam samtools sort -o noHSAligned.out_sorted.bam -T noHS_temp noHSAligned.out.bam samtools index noHSAligned.out_sorted.bam

samtools view -bS 16hrAligned.out.sam > 16hrAligned.out.bam samtools sort -o 16hrAligned.out_sorted.bam -T 16hr_temp 16hrAligned.out.bam samtools index 16hrAligned.out_sorted.bam

rm *Aligned.out.bam

echo "All files are now converted and sorted !"

Is there anything I should add to this script to ensure the files get sorted correctly?

Thank you! Hannah

-- Hannah Vicars, PhD (she/her) Postdoctoral Scholar, Fuller Lab Stanford University

From: Qingqing Wang @.> Date: Wednesday, February 28, 2024 at 8:10 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

Hi,

Did you sort the bam files after STAR mapping?

Convert and sort the resulted alignment files (please use the exact naming nomenclature as shown below). Here we show ctrl 1 sample as an example but users need to do this for all the samples:

$ samtools view -bS ctrl1Aligned.out.sam > ctrl1Aligned.out.bam $ samtools sort -o ctrl1Aligned.out_sorted.bam -T ctrl1_temp ctrl1Aligned.out.bam $ samtools index ctrl1Aligned.out_sorted.bam $ rm *Aligned.out.bam

Qingqing

On Wed, Feb 28, 2024 at 5:55 PM hvicars @.***> wrote:

Hi,

I keep encountering an error while running JUM step A. I am using JUM 3.0.0. The error is: "ERROR: Sort order was unspecified, and file treat1Aligned.out.sorted.bed is not sorted lexicographically. Please rerun with the -g option for a genome file. See documentation for details."

The error appears after line 238. I did STAR mapping for my control and treatment RNA seq data sets before running JUM step A.

Please let me know if there is any other information you need from me or if you have any idea how I might be able to fix this error.

Thank you so much!

— Reply to this email directly, view it on GitHub https://github.com/qqwang-berkeley/JUM/issues/52, or unsubscribe <

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qqwang-berkeley commented 5 months ago

Looks fine to me as well.

Maybe try two more things:

1) could you show me the commands you used to index the genome as well as aligning the reads?

2) could you try sorting the bam files again and run JUM_A one more time?

On Wed, Mar 6, 2024 at 12:57 PM hvicars @.***> wrote:

For STAR mapping, I used:

STAR/2.7.10b samtools/1.17

For JUM step A, I used:

perl/5.34.1 samtools/1.19 bedtools/2.27.1 r/4.0 star/2.7.10b

From: Qingqing Wang @.> Date: Tuesday, March 5, 2024 at 6:23 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

That is really bizarre - it should work then if you aligned the reads against the same indexed genome and sorted them properly.

Which version of bedtools and samtools did you use?

On Mon, Mar 4, 2024 at 4:29 PM hvicars @.***> wrote:

The sequencing length of my read fastq files is 101 bases. Yes, all reads are of equal length.

Hannah Vicars, PhD (she/her) Postdoctoral Scholar, Fuller Lab Stanford University

From: Qingqing Wang @.> Sent: Sunday, March 3, 2024 8:06:56 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>; Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

OK. What is the sequencing length of your read fastq file? Are all the sequencing reads of equal length?

On Fri, Mar 1, 2024 at 2:23 PM hvicars @.***> wrote:

Hi Qingqing,

Yes, I used the script below to convert and sort the alignment files.

Adding module

module add samtools/1.17

samtools view -bS noHSAligned.out.sam > noHSAligned.out.bam samtools sort -o noHSAligned.out_sorted.bam -T noHS_temp noHSAligned.out.bam samtools index noHSAligned.out_sorted.bam

samtools view -bS 16hrAligned.out.sam > 16hrAligned.out.bam samtools sort -o 16hrAligned.out_sorted.bam -T 16hr_temp 16hrAligned.out.bam samtools index 16hrAligned.out_sorted.bam

rm *Aligned.out.bam

echo "All files are now converted and sorted !"

Is there anything I should add to this script to ensure the files get sorted correctly?

Thank you! Hannah

-- Hannah Vicars, PhD (she/her) Postdoctoral Scholar, Fuller Lab Stanford University

From: Qingqing Wang @.> Date: Wednesday, February 28, 2024 at 8:10 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

Hi,

Did you sort the bam files after STAR mapping?

Convert and sort the resulted alignment files (please use the exact naming nomenclature as shown below). Here we show ctrl 1 sample as an example but users need to do this for all the samples:

$ samtools view -bS ctrl1Aligned.out.sam > ctrl1Aligned.out.bam $ samtools sort -o ctrl1Aligned.out_sorted.bam -T ctrl1_temp ctrl1Aligned.out.bam $ samtools index ctrl1Aligned.out_sorted.bam $ rm *Aligned.out.bam

Qingqing

On Wed, Feb 28, 2024 at 5:55 PM hvicars @.***> wrote:

Hi,

I keep encountering an error while running JUM step A. I am using JUM 3.0.0. The error is: "ERROR: Sort order was unspecified, and file treat1Aligned.out.sorted.bed is not sorted lexicographically. Please rerun with the -g option for a genome file. See documentation for details."

The error appears after line 238. I did STAR mapping for my control and treatment RNA seq data sets before running JUM step A.

Please let me know if there is any other information you need from me or if you have any idea how I might be able to fix this error.

Thank you so much!

— Reply to this email directly, view it on GitHub https://github.com/qqwang-berkeley/JUM/issues/52, or unsubscribe <

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hvicars commented 5 months ago

I used the following commands to index the genome:

mkdir genome_index_STAR_r2

STAR --runThreadN 3 \ --runMode genomeGenerate \ --genomeDir genome_index_STAR_r2 \ --genomeFastaFiles Drosophila_melanogaster.BDGP6.32.dna.toplevel.fa \ --sjdbGTFfile Drosophila_melanogaster.BDGP6.32.109.gtf \ --sjdbOverhang 99

I tried sorting the bam files again and running JUM_A, but I still got the same error.

Thank you! Hannah

From: Qingqing Wang @.> Date: Wednesday, March 6, 2024 at 3:55 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue #52) Looks fine to me as well.

Maybe try two more things:

1) could you show me the commands you used to index the genome as well as aligning the reads?

2) could you try sorting the bam files again and run JUM_A one more time?

On Wed, Mar 6, 2024 at 12:57 PM hvicars @.***> wrote:

For STAR mapping, I used:

STAR/2.7.10b samtools/1.17

For JUM step A, I used:

perl/5.34.1 samtools/1.19 bedtools/2.27.1 r/4.0 star/2.7.10b

From: Qingqing Wang @.> Date: Tuesday, March 5, 2024 at 6:23 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

That is really bizarre - it should work then if you aligned the reads against the same indexed genome and sorted them properly.

Which version of bedtools and samtools did you use?

On Mon, Mar 4, 2024 at 4:29 PM hvicars @.***> wrote:

The sequencing length of my read fastq files is 101 bases. Yes, all reads are of equal length.

Hannah Vicars, PhD (she/her) Postdoctoral Scholar, Fuller Lab Stanford University

From: Qingqing Wang @.> Sent: Sunday, March 3, 2024 8:06:56 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>; Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

OK. What is the sequencing length of your read fastq file? Are all the sequencing reads of equal length?

On Fri, Mar 1, 2024 at 2:23 PM hvicars @.***> wrote:

Hi Qingqing,

Yes, I used the script below to convert and sort the alignment files.

Adding module

module add samtools/1.17

samtools view -bS noHSAligned.out.sam > noHSAligned.out.bam samtools sort -o noHSAligned.out_sorted.bam -T noHS_temp noHSAligned.out.bam samtools index noHSAligned.out_sorted.bam

samtools view -bS 16hrAligned.out.sam > 16hrAligned.out.bam samtools sort -o 16hrAligned.out_sorted.bam -T 16hr_temp 16hrAligned.out.bam samtools index 16hrAligned.out_sorted.bam

rm *Aligned.out.bam

echo "All files are now converted and sorted !"

Is there anything I should add to this script to ensure the files get sorted correctly?

Thank you! Hannah

-- Hannah Vicars, PhD (she/her) Postdoctoral Scholar, Fuller Lab Stanford University

From: Qingqing Wang @.> Date: Wednesday, February 28, 2024 at 8:10 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

Hi,

Did you sort the bam files after STAR mapping?

Convert and sort the resulted alignment files (please use the exact naming nomenclature as shown below). Here we show ctrl 1 sample as an example but users need to do this for all the samples:

$ samtools view -bS ctrl1Aligned.out.sam > ctrl1Aligned.out.bam $ samtools sort -o ctrl1Aligned.out_sorted.bam -T ctrl1_temp ctrl1Aligned.out.bam $ samtools index ctrl1Aligned.out_sorted.bam $ rm *Aligned.out.bam

Qingqing

On Wed, Feb 28, 2024 at 5:55 PM hvicars @.***> wrote:

Hi,

I keep encountering an error while running JUM step A. I am using JUM 3.0.0. The error is: "ERROR: Sort order was unspecified, and file treat1Aligned.out.sorted.bed is not sorted lexicographically. Please rerun with the -g option for a genome file. See documentation for details."

The error appears after line 238. I did STAR mapping for my control and treatment RNA seq data sets before running JUM step A.

Please let me know if there is any other information you need from me or if you have any idea how I might be able to fix this error.

Thank you so much!

— Reply to this email directly, view it on GitHub https://github.com/qqwang-berkeley/JUM/issues/52, or unsubscribe <

https://github.com/notifications/unsubscribe-auth/AGJ6PW6HL3YQOY2GLPUGXQDYV6YVHAVCNFSM6AAAAABD64RFQOVHI2DSMVQWIX3LMV43ASLTON2WKOZSGE3DAMBRGQ4TGNA>

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qqwang-berkeley commented 5 months ago

Ok I see. The offending line of code was later than processing the alignment files then. Could you please help me do the following:

In the directory where you ran JUM_A, could you type:

ls -l -t -r

And send me the computer’s response? This is to check the output files going through JUM A code one by one.

Thank you.

On Fri, Mar 8, 2024 at 2:01 PM hvicars @.***> wrote:

I used the following commands to index the genome:

mkdir genome_index_STAR_r2

STAR --runThreadN 3 \ --runMode genomeGenerate \ --genomeDir genome_index_STAR_r2 \ --genomeFastaFiles Drosophila_melanogaster.BDGP6.32.dna.toplevel.fa \ --sjdbGTFfile Drosophila_melanogaster.BDGP6.32.109.gtf \ --sjdbOverhang 99

I tried sorting the bam files again and running JUM_A, but I still got the same error.

Thank you! Hannah

From: Qingqing Wang @.> Date: Wednesday, March 6, 2024 at 3:55 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

Looks fine to me as well.

Maybe try two more things:

1) could you show me the commands you used to index the genome as well as aligning the reads?

2) could you try sorting the bam files again and run JUM_A one more time?

On Wed, Mar 6, 2024 at 12:57 PM hvicars @.***> wrote:

For STAR mapping, I used:

STAR/2.7.10b samtools/1.17

For JUM step A, I used:

perl/5.34.1 samtools/1.19 bedtools/2.27.1 r/4.0 star/2.7.10b

From: Qingqing Wang @.> Date: Tuesday, March 5, 2024 at 6:23 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

That is really bizarre - it should work then if you aligned the reads against the same indexed genome and sorted them properly.

Which version of bedtools and samtools did you use?

On Mon, Mar 4, 2024 at 4:29 PM hvicars @.***> wrote:

The sequencing length of my read fastq files is 101 bases. Yes, all reads are of equal length.

Hannah Vicars, PhD (she/her) Postdoctoral Scholar, Fuller Lab Stanford University

From: Qingqing Wang @.> Sent: Sunday, March 3, 2024 8:06:56 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>; Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

OK. What is the sequencing length of your read fastq file? Are all the sequencing reads of equal length?

On Fri, Mar 1, 2024 at 2:23 PM hvicars @.***> wrote:

Hi Qingqing,

Yes, I used the script below to convert and sort the alignment files.

Adding module

module add samtools/1.17

samtools view -bS noHSAligned.out.sam > noHSAligned.out.bam samtools sort -o noHSAligned.out_sorted.bam -T noHS_temp noHSAligned.out.bam samtools index noHSAligned.out_sorted.bam

samtools view -bS 16hrAligned.out.sam > 16hrAligned.out.bam samtools sort -o 16hrAligned.out_sorted.bam -T 16hr_temp 16hrAligned.out.bam samtools index 16hrAligned.out_sorted.bam

rm *Aligned.out.bam

echo "All files are now converted and sorted !"

Is there anything I should add to this script to ensure the files get sorted correctly?

Thank you! Hannah

-- Hannah Vicars, PhD (she/her) Postdoctoral Scholar, Fuller Lab Stanford University

From: Qingqing Wang @.> Date: Wednesday, February 28, 2024 at 8:10 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

Hi,

Did you sort the bam files after STAR mapping?

Convert and sort the resulted alignment files (please use the exact naming nomenclature as shown below). Here we show ctrl 1 sample as an example but users need to do this for all the samples:

$ samtools view -bS ctrl1Aligned.out.sam > ctrl1Aligned.out.bam $ samtools sort -o ctrl1Aligned.out_sorted.bam -T ctrl1_temp ctrl1Aligned.out.bam $ samtools index ctrl1Aligned.out_sorted.bam $ rm *Aligned.out.bam

Qingqing

On Wed, Feb 28, 2024 at 5:55 PM hvicars @.***> wrote:

Hi,

I keep encountering an error while running JUM step A. I am using JUM 3.0.0. The error is: "ERROR: Sort order was unspecified, and file treat1Aligned.out.sorted.bed is not sorted lexicographically. Please rerun with the -g option for a genome file. See documentation for details."

The error appears after line 238. I did STAR mapping for my control and treatment RNA seq data sets before running JUM step A.

Please let me know if there is any other information you need from me or if you have any idea how I might be able to fix this error.

Thank you so much!

— Reply to this email directly, view it on GitHub https://github.com/qqwang-berkeley/JUM/issues/52, or unsubscribe <

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hvicars commented 5 months ago

In the folder where I ran JUM_A, this is what resulted when I typed ls -l -t -r:

total 21132596

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1271 Feb 28 12:42 slurm-42629967.out

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1273 Feb 28 12:58 slurm-42630797.out

-rw-rw----+ 1 hvicars scg_lab_mtfuller 2148 Feb 28 13:10 slurm-42631308.out

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1582 Feb 28 13:45 slurm-42631718.out

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1939643 Feb 28 14:16 16hrSJ.out.tab_strand_symbol_scaled

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1266241 Feb 28 14:16 16hr_SJ_coor.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 2703283 Feb 28 14:16 UNION_junc_coor_with_junction_ID.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1613719 Feb 28 14:16 UNION_junc_coor.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 2066100 Feb 28 14:16 noHSSJ.out.tab_strand_symbol_scaled

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1347469 Feb 28 14:16 noHS_SJ_coor.txt

drwxrws---+ 2 hvicars scg_lab_mtfuller 4096 Feb 28 14:16 con1_1

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1150960 Feb 28 14:16 UNION_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1264687 Feb 28 14:16 UNION_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted_with_junction_length.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1150960 Feb 28 14:16 UNION_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 2607999 Feb 28 14:16 noHS_junction_counts.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1064334 Feb 28 14:16 condition2_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1125266 Feb 28 14:16 condition1_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted.txt

drwxrws---+ 2 hvicars scg_lab_mtfuller 4096 Feb 28 14:16 con2_1

-rw-rw----+ 1 hvicars scg_lab_mtfuller 2449291 Feb 28 14:16 16hr_junction_counts.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 463661 Feb 28 14:16 UNION_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted_junction_list.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 19964 Feb 28 14:16 more_than_10_union_junc_coor_5_prime_ss_list_with_alternative_3_ss.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 19807 Feb 28 14:16 more_than_10_union_junc_coor_3_prime_ss_list_with_alternative_5_ss.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 389328 Feb 28 14:16 more_than_10_profiled_total_AS_event_junction.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 167491 Feb 28 14:16 more_than_10_profiled_total_AS_event_junction_second_processing_for_JUM_reference_building.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1533426 Feb 28 14:16 more_than_10_profiled_total_AS_event_junction_JUM_annotation.gff

-rw-rw----+ 1 hvicars scg_lab_mtfuller 415284 Feb 28 14:16 more_than_10_profiled_total_AS_event_junction_first_processing_for_JUM_reference_building.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 190299 Feb 28 14:16 more_than_10_profiled_5_ss_and_corresponding_alternative_3_ss_junction_list.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 199029 Feb 28 14:16 more_than_10_profiled_3_ss_and_corresponding_alternative_5_ss_junction_list.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 293881 Feb 28 14:16 noHS_fn_count.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 292773 Feb 28 14:16 16hr_fn_count.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 2574380918 Feb 28 14:19 16hr_temp1

-rw-rw----+ 1 hvicars scg_lab_mtfuller 59731 Feb 28 14:19 16hr_sam_header

-rw-rw----+ 1 hvicars scg_lab_mtfuller 2574440649 Feb 28 14:19 16hr_Aligned.out.spanning_junction_reads.sam

-rw-rw----+ 1 hvicars scg_lab_mtfuller 3273211789 Feb 28 14:19 noHS_temp1

-rw-rw----+ 1 hvicars scg_lab_mtfuller 59739 Feb 28 14:19 noHS_sam_header

-rw-rw----+ 1 hvicars scg_lab_mtfuller 3273271528 Feb 28 14:20 noHS_Aligned.out.spanning_junction_reads.sam

-rw-rw----+ 1 hvicars scg_lab_mtfuller 866324224 Feb 28 14:23 16hr_Aligned.out.spanning_junction_reads.bam

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1111419624 Feb 28 14:24 noHS_Aligned.out.spanning_junction_reads.bam

-rw-rw----+ 1 hvicars scg_lab_mtfuller 854182402 Feb 28 14:25 16hr_Aligned.out.spanning_junction_reads.bed

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1982333 Feb 28 14:26 profiled_16hr_Aligned.out.spanning_junction_reads_junction_overhang_mapped_num.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 0 Feb 28 14:26 16hr_trash_out

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1346305 Feb 28 14:26 16hr_junction_counts_more_than_10_in_all_samples.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1639300 Feb 28 14:26 16hr_junction_counts_more_than_10_in_all_samples_with_both_overhangs.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1087446640 Feb 28 14:26 noHS_Aligned.out.spanning_junction_reads.bed

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1451950 Feb 28 14:27 UNION_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted_with_junction_length_and_overhang_union_from_all_samples.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 2036740 Feb 28 14:27 profiled_noHS_Aligned.out.spanning_junction_reads_junction_overhang_mapped_num.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 0 Feb 28 14:27 noHS_trash_out

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1648222 Feb 28 14:27 noHS_junction_counts_more_than_10_in_all_samples_with_both_overhangs.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1351492 Feb 28 14:27 noHS_junction_counts_more_than_10_in_all_samples.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1324423 Feb 28 14:27 output_short_intron.gff

-rw-rw----+ 1 hvicars scg_lab_mtfuller 2142825 Feb 28 14:27 output_long_intron.gff

-rw-rw----+ 1 hvicars scg_lab_mtfuller 0 Feb 28 14:27 output_short_intron2_sorted.gff

-rw-rw----+ 1 hvicars scg_lab_mtfuller 0 Feb 28 14:27 output_short_intron2.gff

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1324423 Feb 28 14:27 output_short_intron1_sorted.gff

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1324423 Feb 28 14:27 output_short_intron1.gff

-rw-rw----+ 1 hvicars scg_lab_mtfuller 2142825 Feb 28 14:27 output_long_intron_sorted.gff

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1124213 Feb 28 14:27 output_long_intron_adjusted_range_sorted.bed

-rw-rw----+ 1 hvicars scg_lab_mtfuller 0 Feb 28 14:27 output_short_intron_2_adjusted_range_sorted.bed

-rw-rw----+ 1 hvicars scg_lab_mtfuller 678802 Feb 28 14:27 output_short_intron_1_adjusted_range_sorted.bed

-rw-rw----+ 1 hvicars scg_lab_mtfuller 2964869000 Feb 28 14:29 16hrAligned.out.bed

-rw-rw----+ 1 hvicars scg_lab_mtfuller 2964869000 Feb 28 14:39 16hrAligned.out.sorted.bed

-rw-rw----+ 1 hvicars scg_lab_mtfuller 47235833 Feb 28 14:40 16hrAligned.out_intersect_long_intron.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1582 Feb 28 14:40 slurm-42634399.out

-rw-rw----+ 1 hvicars scg_lab_mtfuller 577060 Feb 28 14:40 16hrAligned.out_intersect_short_intron1.txt

-rw-rw----+ 1 hvicars scg_lab_mtfuller 1070 Mar 1 11:18 JUM_stepA_nohs_v_16hr.sh

Let me know what else you need me to do. Thanks!

Hannah

From: Qingqing Wang @.> Date: Monday, March 11, 2024 at 6:02 AM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue #52) Ok I see. The offending line of code was later than processing the alignment files then. Could you please help me do the following:

In the directory where you ran JUM_A, could you type:

ls -l -t -r

And send me the computer’s response? This is to check the output files going through JUM A code one by one.

Thank you.

On Fri, Mar 8, 2024 at 2:01 PM hvicars @.***> wrote:

I used the following commands to index the genome:

mkdir genome_index_STAR_r2

STAR --runThreadN 3 \ --runMode genomeGenerate \ --genomeDir genome_index_STAR_r2 \ --genomeFastaFiles Drosophila_melanogaster.BDGP6.32.dna.toplevel.fa \ --sjdbGTFfile Drosophila_melanogaster.BDGP6.32.109.gtf \ --sjdbOverhang 99

I tried sorting the bam files again and running JUM_A, but I still got the same error.

Thank you! Hannah

From: Qingqing Wang @.> Date: Wednesday, March 6, 2024 at 3:55 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

Looks fine to me as well.

Maybe try two more things:

1) could you show me the commands you used to index the genome as well as aligning the reads?

2) could you try sorting the bam files again and run JUM_A one more time?

On Wed, Mar 6, 2024 at 12:57 PM hvicars @.***> wrote:

For STAR mapping, I used:

STAR/2.7.10b samtools/1.17

For JUM step A, I used:

perl/5.34.1 samtools/1.19 bedtools/2.27.1 r/4.0 star/2.7.10b

From: Qingqing Wang @.> Date: Tuesday, March 5, 2024 at 6:23 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

That is really bizarre - it should work then if you aligned the reads against the same indexed genome and sorted them properly.

Which version of bedtools and samtools did you use?

On Mon, Mar 4, 2024 at 4:29 PM hvicars @.***> wrote:

The sequencing length of my read fastq files is 101 bases. Yes, all reads are of equal length.

Hannah Vicars, PhD (she/her) Postdoctoral Scholar, Fuller Lab Stanford University

From: Qingqing Wang @.> Sent: Sunday, March 3, 2024 8:06:56 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>; Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

OK. What is the sequencing length of your read fastq file? Are all the sequencing reads of equal length?

On Fri, Mar 1, 2024 at 2:23 PM hvicars @.***> wrote:

Hi Qingqing,

Yes, I used the script below to convert and sort the alignment files.

Adding module

module add samtools/1.17

samtools view -bS noHSAligned.out.sam > noHSAligned.out.bam samtools sort -o noHSAligned.out_sorted.bam -T noHS_temp noHSAligned.out.bam samtools index noHSAligned.out_sorted.bam

samtools view -bS 16hrAligned.out.sam > 16hrAligned.out.bam samtools sort -o 16hrAligned.out_sorted.bam -T 16hr_temp 16hrAligned.out.bam samtools index 16hrAligned.out_sorted.bam

rm *Aligned.out.bam

echo "All files are now converted and sorted !"

Is there anything I should add to this script to ensure the files get sorted correctly?

Thank you! Hannah

-- Hannah Vicars, PhD (she/her) Postdoctoral Scholar, Fuller Lab Stanford University

From: Qingqing Wang @.> Date: Wednesday, February 28, 2024 at 8:10 PM To: qqwang-berkeley/JUM @.> Cc: Hannah Marie Vicars @.>, Author @.> Subject: Re: [qqwang-berkeley/JUM] Error when running JUM step A (Issue

52)

Hi,

Did you sort the bam files after STAR mapping?

Convert and sort the resulted alignment files (please use the exact naming nomenclature as shown below). Here we show ctrl 1 sample as an example but users need to do this for all the samples:

$ samtools view -bS ctrl1Aligned.out.sam > ctrl1Aligned.out.bam $ samtools sort -o ctrl1Aligned.out_sorted.bam -T ctrl1_temp ctrl1Aligned.out.bam $ samtools index ctrl1Aligned.out_sorted.bam $ rm *Aligned.out.bam

Qingqing

On Wed, Feb 28, 2024 at 5:55 PM hvicars @.***> wrote:

Hi,

I keep encountering an error while running JUM step A. I am using JUM 3.0.0. The error is: "ERROR: Sort order was unspecified, and file treat1Aligned.out.sorted.bed is not sorted lexicographically. Please rerun with the -g option for a genome file. See documentation for details."

The error appears after line 238. I did STAR mapping for my control and treatment RNA seq data sets before running JUM step A.

Please let me know if there is any other information you need from me or if you have any idea how I might be able to fix this error.

Thank you so much!

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hvicars commented 4 months ago

Hi,

I re-indexed my genome and re-ran JUM_A after STAR mapping and received the same result. I then typed "ls -l -t -r" in the folder where I redid JUM_A, and this was my result:

total 57129220 -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller drwxrws---+ 2 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller drwxrws---+ 2 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller -rw-rw----+ 1 hvicars scg_lab_mtfuller 16782164691 Apr 3 12:24 noHSAligned.out.sam 2066100 Apr 3 12:24 noHSSJ.out.tab 13542666415 Apr 4 12:21 16hrAligned.out.sam 1939643 Apr 4 12:22 16hrSJ.out.tab 3589751512 Apr 4 13:29 noHSAligned.out_sorted.bam 2941872065 Apr 4 13:56 16hrAligned.out_sorted.bam 1135 Apr 8 10:08 slurm-42826020.out 1021 Apr 8 10:10 JUM_stepA_nohs_v_16hr.sh 1939643 Apr 8 10:12 16hrSJ.out.tab_strand_symbol_scaled 1266241 Apr 8 10:12 16hr_SJ_coor.txt 2703283 Apr 8 10:12 UNION_junc_coor_with_junction_ID.txt 1613719 Apr 8 10:12 UNION_junc_coor.txt 2066100 Apr 8 10:12 noHSSJ.out.tab_strand_symbol_scaled 1347469 Apr 8 10:12 noHS_SJ_coor.txt 4096 Apr 8 10:12 con1_1 1150960 Apr 8 10:12 UNION_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples.txt 1264687 Apr 8 10:12 UNION_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted_with_junction_length.txt 1150960 Apr 8 10:12 UNION_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted.txt 2607999 Apr 8 10:12 noHS_junction_counts.txt 1064334 Apr 8 10:12 condition2_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted.txt 1125266 Apr 8 10:12 condition1_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted.txt 4096 Apr 8 10:12 con2_1 2449291 Apr 8 10:12 16hr_junction_counts.txt 463661 Apr 8 10:12 UNION_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted_junction_list.txt 19964 Apr 8 10:12 more_than_10_union_junc_coor_5_prime_ss_list_with_alternative_3_ss.txt 19807 Apr 8 10:12 more_than_10_union_junc_coor_3_prime_ss_list_with_alternative_5_ss.txt 389328 Apr 8 10:12 more_than_10_profiled_total_AS_event_junction.txt 167491 Apr 8 10:12 more_than_10_profiled_total_AS_event_junction_second_processing_for_JUM_reference_building.txt 1533426 Apr 8 10:12 more_than_10_profiled_total_AS_event_junction_JUM_annotation.gff 415284 Apr 8 10:12 more_than_10_profiled_total_AS_event_junction_first_processing_for_JUM_reference_building.txt 190299 Apr 8 10:12 more_than_10_profiled_5_ss_and_corresponding_alternative_3_ss_junction_list.txt 199029 Apr 8 10:12 more_than_10_profiled_3_ss_and_corresponding_alternative_5_ss_junction_list.txt 293881 Apr 8 10:12 noHS_fn_count.txt 292773 Apr 8 10:12 16hr_fn_count.txt 2574382249 Apr 8 10:14 16hr_temp1 59731 Apr 8 10:14 16hr_sam_header 2574441980 Apr 8 10:14 16hr_Aligned.out.spanning_junction_reads.sam 3273212119 Apr 8 10:15 noHS_temp1 59739 Apr 8 10:15 noHS_sam_header 3273271858 Apr 8 10:15 noHS_Aligned.out.spanning_junction_reads.sam 866329467 Apr 8 10:18 16hr_Aligned.out.spanning_junction_reads.bam 1111440379 Apr 8 10:19 noHS_Aligned.out.spanning_junction_reads.bam 854182853 Apr 8 10:20 16hr_Aligned.out.spanning_junction_reads.bed 1982414 Apr 8 10:21 profiled_16hr_Aligned.out.spanning_junction_reads_junction_overhang_mapped_num.txt 0 Apr 8 10:21 16hr_trash_out 1639300 Apr 8 10:21 16hr_junction_counts_more_than_10_in_all_samples_with_both_overhangs.txt 1346305 Apr 8 10:21 16hr_junction_counts_more_than_10_in_all_samples.txt 1087446746 Apr 8 10:21 noHS_Aligned.out.spanning_junction_reads.bed 2036740 Apr 8 10:22 profiled_noHS_Aligned.out.spanning_junction_reads_junction_overhang_mapped_num.txt 0 Apr 8 10:22 noHS_trash_out 1351492 Apr 8 10:22 noHS_junction_counts_more_than_10_in_all_samples.txt 1451950 Apr 8 10:22 UNION_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted_with_junction_length_and_overhang_union_from_all_samples.txt 1324423 Apr 8 10:22 output_short_intron.gff 2142825 Apr 8 10:22 output_long_intron.gff 1648222 Apr 8 10:22 noHS_junction_counts_more_than_10_in_all_samples_with_both_overhangs.txt 0 Apr 8 10:22 output_short_intron2.gff 1324423 Apr 8 10:22 output_short_intron1.gff 2142825 Apr 8 10:22 output_long_intron_sorted.gff 0 Apr 8 10:22 output_short_intron2_sorted.gff 0 Apr 8 10:22 output_short_intron_2_adjusted_range_sorted.bed 1324423 Apr 8 10:22 output_short_intron1_sorted.gff 678802 Apr 8 10:22 output_short_intron_1_adjusted_range_sorted.bed 1124213 Apr 8 10:22 output_long_intron_adjusted_range_sorted.bed 2964878810 Apr 8 10:24 16hrAligned.out.bed 2964878810 Apr 8 10:34 16hrAligned.out.sorted.bed 47240647 Apr 8 10:35 16hrAligned.out_intersect_long_intron.txt 1581 Apr 8 10:35 slurm-42826023.out 577060 Apr 8 10:35 16hrAligned.out_intersect_short_intron1.txt

Please let me know if this helps at all or if there's anything more I can do to narrow down this issue. Would it be better if I used an older version of JUM?

Thanks! Hannah

qqwang-berkeley commented 4 months ago

Can you run the following two commands one by one in the directory where you ran JUM and let me know what the response is? I think that is the offending step but I want to confirm.

bedtools bamtobed -i noHSAligned.out_sorted.bam > noHSAligned.out.bed sort -k1,1 -k2,2n noHSAligned.out.bed > noHSAligned.out.sorted.bed

On Thu, Apr 11, 2024 at 12:48 PM hvicars @.***> wrote:

Hi,

I re-indexed my genome and re-ran JUM_A after STAR mapping and received the same result. I then typed "ls -l -t -r" in the folder where I redid JUM_A, and this was my result:

total 57129220 -rw-rw----+ 1 hvicars scg_lab_mtfuller 16782164691 Apr 3 12:24 noHSAligned.out.sam -rw-rw----+ 1 hvicars scg_lab_mtfuller 2066100 Apr 3 12:24 noHSSJ.out.tab -rw-rw----+ 1 hvicars scg_lab_mtfuller 13542666415 Apr 4 12:21 16hrAligned.out.sam -rw-rw----+ 1 hvicars scg_lab_mtfuller 1939643 Apr 4 12:22 16hrSJ.out.tab -rw-rw----+ 1 hvicars scg_lab_mtfuller 3589751512 Apr 4 13:29 noHSAligned.out_sorted.bam -rw-rw----+ 1 hvicars scg_lab_mtfuller 2941872065 Apr 4 13:56 16hrAligned.out_sorted.bam -rw-rw----+ 1 hvicars scg_lab_mtfuller 1135 Apr 8 10:08 slurm-42826020.out -rw-rw----+ 1 hvicars scg_lab_mtfuller 1021 Apr 8 10:10 JUM_stepA_nohs_v_16hr.sh -rw-rw----+ 1 hvicars scg_lab_mtfuller 1939643 Apr 8 10:12 16hrSJ.out.tab_strand_symbol_scaled -rw-rw----+ 1 hvicars scg_lab_mtfuller 1266241 Apr 8 10:12 16hr_SJ_coor.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 2703283 Apr 8 10:12 UNION_junc_coor_with_junction_ID.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 1613719 Apr 8 10:12 UNION_junc_coor.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 2066100 Apr 8 10:12 noHSSJ.out.tab_strand_symbol_scaled -rw-rw----+ 1 hvicars scg_lab_mtfuller 1347469 Apr 8 10:12 noHS_SJ_coor.txt drwxrws---+ 2 hvicars scg_lab_mtfuller 4096 Apr 8 10:12 con1_1 -rw-rw----+ 1 hvicars scg_lab_mtfuller 1150960 Apr 8 10:12 UNION_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 1264687 Apr 8 10:12 UNION_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted_with_junction_length.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 1150960 Apr 8 10:12 UNION_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 2607999 Apr 8 10:12 noHS_junction_counts.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 1064334 Apr 8 10:12 condition2_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 1125266 Apr 8 10:12 condition1_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted.txt drwxrws---+ 2 hvicars scg_lab_mtfuller 4096 Apr 8 10:12 con2_1 -rw-rw----+ 1 hvicars scg_lab_mtfuller 2449291 Apr 8 10:12 16hr_junction_counts.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 463661 Apr 8 10:12 UNION_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted_junction_list.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 19964 Apr 8 10:12 more_than_10_union_junc_coor_5_prime_ss_list_with_alternative_3_ss.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 19807 Apr 8 10:12 more_than_10_union_junc_coor_3_prime_ss_list_with_alternative_5_ss.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 389328 Apr 8 10:12 more_than_10_profiled_total_AS_event_junction.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 167491 Apr 8 10:12 more_than_10_profiled_total_AS_event_junction_second_processing_for_JUM_reference_building.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 1533426 Apr 8 10:12 more_than_10_profiled_total_AS_event_junction_JUM_annotation.gff -rw-rw----+ 1 hvicars scg_lab_mtfuller 415284 Apr 8 10:12 more_than_10_profiled_total_AS_event_junction_first_processing_for_JUM_reference_building.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 190299 Apr 8 10:12 more_than_10_profiled_5_ss_and_corresponding_alternative_3_ss_junction_list.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 199029 Apr 8 10:12 more_than_10_profiled_3_ss_and_corresponding_alternative_5_ss_junction_list.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 293881 Apr 8 10:12 noHS_fn_count.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 292773 Apr 8 10:12 16hr_fn_count.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 2574382249 Apr 8 10:14 16hr_temp1 -rw-rw----+ 1 hvicars scg_lab_mtfuller 59731 Apr 8 10:14 16hr_sam_header -rw-rw----+ 1 hvicars scg_lab_mtfuller 2574441980 Apr 8 10:14 16hr_Aligned.out.spanning_junction_reads.sam -rw-rw----+ 1 hvicars scg_lab_mtfuller 3273212119 Apr 8 10:15 noHS_temp1 -rw-rw----+ 1 hvicars scg_lab_mtfuller 59739 Apr 8 10:15 noHS_sam_header -rw-rw----+ 1 hvicars scg_lab_mtfuller 3273271858 Apr 8 10:15 noHS_Aligned.out.spanning_junction_reads.sam -rw-rw----+ 1 hvicars scg_lab_mtfuller 866329467 Apr 8 10:18 16hr_Aligned.out.spanning_junction_reads.bam -rw-rw----+ 1 hvicars scg_lab_mtfuller 1111440379 Apr 8 10:19 noHS_Aligned.out.spanning_junction_reads.bam -rw-rw----+ 1 hvicars scg_lab_mtfuller 854182853 Apr 8 10:20 16hr_Aligned.out.spanning_junction_reads.bed -rw-rw----+ 1 hvicars scg_lab_mtfuller 1982414 Apr 8 10:21 profiled_16hr_Aligned.out.spanning_junction_reads_junction_overhang_mapped_num.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 0 Apr 8 10:21 16hr_trash_out -rw-rw----+ 1 hvicars scg_lab_mtfuller 1639300 Apr 8 10:21 16hr_junction_counts_more_than_10_in_all_samples_with_both_overhangs.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 1346305 Apr 8 10:21 16hr_junction_counts_more_than_10_in_all_samples.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 1087446746 Apr 8 10:21 noHS_Aligned.out.spanning_junction_reads.bed -rw-rw----+ 1 hvicars scg_lab_mtfuller 2036740 Apr 8 10:22 profiled_noHS_Aligned.out.spanning_junction_reads_junction_overhang_mapped_num.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 0 Apr 8 10:22 noHS_trash_out -rw-rw----+ 1 hvicars scg_lab_mtfuller 1351492 Apr 8 10:22 noHS_junction_counts_more_than_10_in_all_samples.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 1451950 Apr 8 10:22 UNION_junc_coor_with_junction_ID_more_than_10_read_in_at_least_1_samples_formatted_with_junction_length_and_overhang_union_from_all_samples.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 1324423 Apr 8 10:22 output_short_intron.gff -rw-rw----+ 1 hvicars scg_lab_mtfuller 2142825 Apr 8 10:22 output_long_intron.gff -rw-rw----+ 1 hvicars scg_lab_mtfuller 1648222 Apr 8 10:22 noHS_junction_counts_more_than_10_in_all_samples_with_both_overhangs.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 0 Apr 8 10:22 output_short_intron2.gff -rw-rw----+ 1 hvicars scg_lab_mtfuller 1324423 Apr 8 10:22 output_short_intron1.gff -rw-rw----+ 1 hvicars scg_lab_mtfuller 2142825 Apr 8 10:22 output_long_intron_sorted.gff -rw-rw----+ 1 hvicars scg_lab_mtfuller 0 Apr 8 10:22 output_short_intron2_sorted.gff -rw-rw----+ 1 hvicars scg_lab_mtfuller 0 Apr 8 10:22 output_short_intron_2_adjusted_range_sorted.bed -rw-rw----+ 1 hvicars scg_lab_mtfuller 1324423 Apr 8 10:22 output_short_intron1_sorted.gff -rw-rw----+ 1 hvicars scg_lab_mtfuller 678802 Apr 8 10:22 output_short_intron_1_adjusted_range_sorted.bed -rw-rw----+ 1 hvicars scg_lab_mtfuller 1124213 Apr 8 10:22 output_long_intron_adjusted_range_sorted.bed -rw-rw----+ 1 hvicars scg_lab_mtfuller 2964878810 Apr 8 10:24 16hrAligned.out.bed -rw-rw----+ 1 hvicars scg_lab_mtfuller 2964878810 Apr 8 10:34 16hrAligned.out.sorted.bed -rw-rw----+ 1 hvicars scg_lab_mtfuller 47240647 Apr 8 10:35 16hrAligned.out_intersect_long_intron.txt -rw-rw----+ 1 hvicars scg_lab_mtfuller 1581 Apr 8 10:35 slurm-42826023.out -rw-rw----+ 1 hvicars scg_lab_mtfuller 577060 Apr 8 10:35 16hrAligned.out_intersect_short_intron1.txt

Please let me know if this helps at all or if there's anything more I can do to narrow down this issue. Would it be better if I used an older version of JUM?

Thanks! Hannah

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hvicars commented 4 months ago

Okay, I ran the two commands in the directory where I ran JUM_A. The outputs were:

noHSAligned.out.sorted.bed noHSAligned.out.bed

What would you like me to do next?

qqwang-berkeley commented 4 months ago

Surprisingly it ran without error. Then it is not the input files at all as they were processed successfully.

Could you help me with three more things?

1) Can you send me the complete output from running JUM_A.sh, including the error line? The offending line may be later than I previously thought.

2) Can you run the following command under the directory that you ran JUM and see if any error message pops out? sort -k1,1 -k4,4n output_short_intron2.gff > output_short_intron2_sorted.gff

3) Can you send me the file "output_short_intron.gff"? It should be only 1.3kb.

On Fri, Apr 12, 2024 at 4:08 PM hvicars @.***> wrote:

Okay, I ran the two commands in the directory where I ran JUM_A. The outputs were:

noHSAligned.out.sorted.bed noHSAligned.out.bed

What would you like me to do next?

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hvicars commented 4 months ago

1) Output from JUM_A.sh: 2024-04-15 14:30:15 (625 KB/s) - written to stdout [302419/302419]

App::cpanminus is up to date. (1.7047) local::lib is up to date. (2.000029) Array::Utils is up to date. (0.5) Statistics::Descriptive is up to date. (3.0801) perl modules successfully loaded Sample names are: noHS 16hr 1 input samples to consider under the current condition 1 input samples to consider under the current condition Preparing for intron retention AS events analyses... Processing junction overhangs for Sample 16hr... Success. Processing junction overhangs for Sample noHS... Success. Line238 Done ERROR: Sort order was unspecified, and file 16hrAligned.out.sorted.bed is not sorted lexicographically. Please rerun with the -g option for a genome file. See documentation for details.

2) This sorting was successful. No error message popped up.

3) However, the following files are empty (zero bytes):

output_short_intron2.gff output_short_intron2_sorted.gff output_short_intron_2_adjusted_range_sorted.bed

Let me know what else you need from me. Thanks!

qqwang-berkeley commented 4 months ago

OK. We are getting closer. I think you got the error because of the empty gff file from intron2. Let's test if my thinking is correct.

Could you run the following commands in the same directory and let me know if you see similar errors you saw from running JUM_A.sh: bedtools intersect -a 16hrAligned.out.sorted.bed -b output_short_intron1_sorted.gff -sorted -wa -u -f 1 > test_short_intron1 bedtools intersect -a 16hrAligned.out.sorted.bed -b output_short_intron2_sorted.gff -sorted -wa -u -F 1 > test_bam_short_intron2

On Tue, Apr 16, 2024 at 5:22 PM hvicars @.***> wrote:

Output from JUM_A.sh: 2024-04-15 14:30:15 (625 KB/s) - written to stdout [302419/302419]

App::cpanminus is up to date. (1.7047) local::lib is up to date. (2.000029) Array::Utils is up to date. (0.5) Statistics::Descriptive is up to date. (3.0801) perl modules successfully loaded Sample names are: noHS 16hr 1 input samples to consider under the current condition 1 input samples to consider under the current condition Preparing for intron retention AS events analyses... Processing junction overhangs for Sample 16hr... Success. Processing junction overhangs for Sample noHS... Success. Line238 Done ERROR: Sort order was unspecified, and file 16hrAligned.out.sorted.bed is not sorted lexicographically. Please rerun with the -g option for a genome file. See documentation for details.

2.

This sorting was successful. No error message popped up. 3.

However, the following files are empty (zero bytes):

output_short_intron2.gff output_short_intron2_sorted.gff output_short_intron_2_adjusted_range_sorted.bed

Let me know what else you need from me. Thanks!

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hvicars commented 4 months ago

This is the output I got from running those commands:

Tool: bedtools intersect (aka intersectBed) Version: v2.27.1 Summary: Report overlaps between two feature files.

Usage: bedtools intersect [OPTIONS] -a <bed/gff/vcf/bam> -b <bed/gff/vcf/bam>

Note: -b may be followed with multiple databases and/or 
wildcard (*) character(s).

Options: -wa Write the original entry in A for each overlap.

-wb Write the original entry in B for each overlap.
    - Useful for knowing _what_ A overlaps. Restricted by -f and -r.

-loj    Perform a "left outer join". That is, for each feature in A
    report each overlap with B.  If no overlaps are found, 
    report a NULL feature for B.

-wo Write the original A and B entries plus the number of base
    pairs of overlap between the two features.
    - Overlaps restricted by -f and -r.
      Only A features with overlap are reported.

-wao    Write the original A and B entries plus the number of base
    pairs of overlap between the two features.
    - Overlapping features restricted by -f and -r.
      However, A features w/o overlap are also reported
      with a NULL B feature and overlap = 0.

-u  Write the original A entry _once_ if _any_ overlaps found in B.
    - In other words, just report the fact >=1 hit was found.
    - Overlaps restricted by -f and -r.

-c  For each entry in A, report the number of overlaps with B.
    - Reports 0 for A entries that have no overlap with B.
    - Overlaps restricted by -f and -r.

-v  Only report those entries in A that have _no overlaps_ with B.
    - Similar to "grep -v" (an homage).

-ubam   Write uncompressed BAM output. Default writes compressed BAM.

-s  Require same strandedness.  That is, only report hits in B
    that overlap A on the _same_ strand.
    - By default, overlaps are reported without respect to strand.

-S  Require different strandedness.  That is, only report hits in B
    that overlap A on the _opposite_ strand.
    - By default, overlaps are reported without respect to strand.

-f  Minimum overlap required as a fraction of A.
    - Default is 1E-9 (i.e., 1bp).
    - FLOAT (e.g. 0.50)

-F  Minimum overlap required as a fraction of B.
    - Default is 1E-9 (i.e., 1bp).
    - FLOAT (e.g. 0.50)

-r  Require that the fraction overlap be reciprocal for A AND B.
    - In other words, if -f is 0.90 and -r is used, this requires
      that B overlap 90% of A and A _also_ overlaps 90% of B.

-e  Require that the minimum fraction be satisfied for A OR B.
    - In other words, if -e is used with -f 0.90 and -F 0.10 this requires
      that either 90% of A is covered OR 10% of  B is covered.
      Without -e, both fractions would have to be satisfied.

-split  Treat "split" BAM or BED12 entries as distinct BED intervals.

-g  Provide a genome file to enforce consistent chromosome sort order
    across input files. Only applies when used with -sorted option.

-nonamecheck    For sorted data, don't throw an error if the file has different naming conventions
        for the same chromosome. ex. "chr1" vs "chr01".

-sorted Use the "chromsweep" algorithm for sorted (-k1,1 -k2,2n) input.

-names  When using multiple databases, provide an alias for each that
    will appear instead of a fileId when also printing the DB record.

-filenames  When using multiple databases, show each complete filename
        instead of a fileId when also printing the DB record.

-sortout    When using multiple databases, sort the output DB hits
        for each record.

-bed    If using BAM input, write output as BED.

-header Print the header from the A file prior to results.

-nobuf  Disable buffered output. Using this option will cause each line
    of output to be printed as it is generated, rather than saved
    in a buffer. This will make printing large output files 
    noticeably slower, but can be useful in conjunction with
    other software tools and scripts that need to process one
    line of bedtools output at a time.

-iobuf  Specify amount of memory to use for input buffer.
    Takes an integer argument. Optional suffixes K/M/G supported.
    Note: currently has no effect with compressed files.

Notes: (1) When a BAM file is used for the A file, the alignment is retained if overlaps exist, and excluded if an overlap cannot be found. If multiple overlaps exist, they are not reported, as we are only testing for one or more overlaps.

ERROR: -b option given, but no database file specified. /var/spool/slurm/slurmd/job42896608/slurm_script: line 12: output_short_intron1_sorted.gff: command not found

Tool: bedtools intersect (aka intersectBed) Version: v2.27.1 Summary: Report overlaps between two feature files.

Usage: bedtools intersect [OPTIONS] -a <bed/gff/vcf/bam> -b <bed/gff/vcf/bam>

Note: -b may be followed with multiple databases and/or 
wildcard (*) character(s).

Options: -wa Write the original entry in A for each overlap.

-wb Write the original entry in B for each overlap.
    - Useful for knowing _what_ A overlaps. Restricted by -f and -r.

-loj    Perform a "left outer join". That is, for each feature in A
    report each overlap with B.  If no overlaps are found, 
    report a NULL feature for B.

-wo Write the original A and B entries plus the number of base
    pairs of overlap between the two features.
    - Overlaps restricted by -f and -r.
      Only A features with overlap are reported.

-wao    Write the original A and B entries plus the number of base
    pairs of overlap between the two features.
    - Overlapping features restricted by -f and -r.
      However, A features w/o overlap are also reported
      with a NULL B feature and overlap = 0.

-u  Write the original A entry _once_ if _any_ overlaps found in B.
    - In other words, just report the fact >=1 hit was found.
    - Overlaps restricted by -f and -r.

-c  For each entry in A, report the number of overlaps with B.
    - Reports 0 for A entries that have no overlap with B.
    - Overlaps restricted by -f and -r.

-v  Only report those entries in A that have _no overlaps_ with B.
    - Similar to "grep -v" (an homage).

-ubam   Write uncompressed BAM output. Default writes compressed BAM.

-s  Require same strandedness.  That is, only report hits in B
    that overlap A on the _same_ strand.
    - By default, overlaps are reported without respect to strand.

-S  Require different strandedness.  That is, only report hits in B
    that overlap A on the _opposite_ strand.
    - By default, overlaps are reported without respect to strand.

-f  Minimum overlap required as a fraction of A.
    - Default is 1E-9 (i.e., 1bp).
    - FLOAT (e.g. 0.50)

-F  Minimum overlap required as a fraction of B.
    - Default is 1E-9 (i.e., 1bp).
    - FLOAT (e.g. 0.50)

-r  Require that the fraction overlap be reciprocal for A AND B.
    - In other words, if -f is 0.90 and -r is used, this requires
      that B overlap 90% of A and A _also_ overlaps 90% of B.

-e  Require that the minimum fraction be satisfied for A OR B.
    - In other words, if -e is used with -f 0.90 and -F 0.10 this requires
      that either 90% of A is covered OR 10% of  B is covered.
      Without -e, both fractions would have to be satisfied.

-split  Treat "split" BAM or BED12 entries as distinct BED intervals.

-g  Provide a genome file to enforce consistent chromosome sort order
    across input files. Only applies when used with -sorted option.

-nonamecheck    For sorted data, don't throw an error if the file has different naming conventions
        for the same chromosome. ex. "chr1" vs "chr01".

-sorted Use the "chromsweep" algorithm for sorted (-k1,1 -k2,2n) input.

-names  When using multiple databases, provide an alias for each that
    will appear instead of a fileId when also printing the DB record.

-filenames  When using multiple databases, show each complete filename
        instead of a fileId when also printing the DB record.

-sortout    When using multiple databases, sort the output DB hits
        for each record.

-bed    If using BAM input, write output as BED.

-header Print the header from the A file prior to results.

-nobuf  Disable buffered output. Using this option will cause each line
    of output to be printed as it is generated, rather than saved
    in a buffer. This will make printing large output files 
    noticeably slower, but can be useful in conjunction with
    other software tools and scripts that need to process one
    line of bedtools output at a time.

-iobuf  Specify amount of memory to use for input buffer.
    Takes an integer argument. Optional suffixes K/M/G supported.
    Note: currently has no effect with compressed files.

ERROR: -b option given, but no database file specified. /var/spool/slurm/slurmd/job42896608/slurm_script: line 14: output_short_intron2_sorted.gff: command not found Troubleshooting is now completed!

The following files were created but are empty (zero bytes): test_bam_short_intron2 test_short_intron1

qqwang-berkeley commented 4 months ago

I don't understand the output of the two commands? Did you run them one after another directly on a terminal? By submitting a slurm job sh file? JupyterHub Node? Galaxy system?

The two files output_short_intron1_sorted.gff and output_short_intron2_sorted.gff are clearly in the directory where you ran JUM_A.sh (as shown by the results of your "ls -l -t -r" command) and I don't understand why the computer said it could not find them. And this output looks like trouble-shooting messages to me rather than direct computer output after receiving the commands?

On Wed, Apr 17, 2024 at 1:47 PM hvicars @.***> wrote:

This is the output I got from running those commands:

Tool: bedtools intersect (aka intersectBed) Version: v2.27.1 Summary: Report overlaps between two feature files.

Usage: bedtools intersect [OPTIONS] -a <bed/gff/vcf/bam> -b <bed/gff/vcf/bam>

Note: -b may be followed with multiple databases and/or wildcard (*) character(s).

Options: -wa Write the original entry in A for each overlap.

-wb Write the original entry in B for each overlap.

Useful for knowing what A overlaps. Restricted by -f and -r.

-loj Perform a "left outer join". That is, for each feature in A report each overlap with B. If no overlaps are found, report a NULL feature for B.

-wo Write the original A and B entries plus the number of base pairs of overlap between the two features.

Overlaps restricted by -f and -r. Only A features with overlap are reported.

-wao Write the original A and B entries plus the number of base pairs of overlap between the two features.

Overlapping features restricted by -f and -r. However, A features w/o overlap are also reported with a NULL B feature and overlap = 0.

-u Write the original A entry once if any overlaps found in B.

In other words, just report the fact >=1 hit was found.

Overlaps restricted by -f and -r.

-c For each entry in A, report the number of overlaps with B.

Reports 0 for A entries that have no overlap with B.

Overlaps restricted by -f and -r.

-v Only report those entries in A that have no overlaps with B.

Similar to "grep -v" (an homage).

-ubam Write uncompressed BAM output. Default writes compressed BAM.

-s Require same strandedness. That is, only report hits in B that overlap A on the same strand.

By default, overlaps are reported without respect to strand.

-S Require different strandedness. That is, only report hits in B that overlap A on the opposite strand.

By default, overlaps are reported without respect to strand.

-f Minimum overlap required as a fraction of A.

Default is 1E-9 (i.e., 1bp).

FLOAT (e.g. 0.50)

-F Minimum overlap required as a fraction of B.

Default is 1E-9 (i.e., 1bp).

FLOAT (e.g. 0.50)

-r Require that the fraction overlap be reciprocal for A AND B.

In other words, if -f is 0.90 and -r is used, this requires that B overlap 90% of A and A also overlaps 90% of B.

-e Require that the minimum fraction be satisfied for A OR B.

In other words, if -e is used with -f 0.90 and -F 0.10 this requires that either 90% of A is covered OR 10% of B is covered. Without -e, both fractions would have to be satisfied.

-split Treat "split" BAM or BED12 entries as distinct BED intervals.

-g Provide a genome file to enforce consistent chromosome sort order across input files. Only applies when used with -sorted option.

-nonamecheck For sorted data, don't throw an error if the file has different naming conventions for the same chromosome. ex. "chr1" vs "chr01".

-sorted Use the "chromsweep" algorithm for sorted (-k1,1 -k2,2n) input.

-names When using multiple databases, provide an alias for each that will appear instead of a fileId when also printing the DB record.

-filenames When using multiple databases, show each complete filename instead of a fileId when also printing the DB record.

-sortout When using multiple databases, sort the output DB hits for each record.

-bed If using BAM input, write output as BED.

-header Print the header from the A file prior to results.

-nobuf Disable buffered output. Using this option will cause each line of output to be printed as it is generated, rather than saved in a buffer. This will make printing large output files noticeably slower, but can be useful in conjunction with other software tools and scripts that need to process one line of bedtools output at a time.

-iobuf Specify amount of memory to use for input buffer. Takes an integer argument. Optional suffixes K/M/G supported. Note: currently has no effect with compressed files.

Notes: (1) When a BAM file is used for the A file, the alignment is retained if overlaps exist, and excluded if an overlap cannot be found. If multiple overlaps exist, they are not reported, as we are only testing for one or more overlaps.

ERROR: -b option given, but no database file specified. /var/spool/slurm/slurmd/job42896608/slurm_script: line 12: output_short_intron1_sorted.gff: command not found

Tool: bedtools intersect (aka intersectBed) Version: v2.27.1 Summary: Report overlaps between two feature files.

Usage: bedtools intersect [OPTIONS] -a <bed/gff/vcf/bam> -b <bed/gff/vcf/bam>

Note: -b may be followed with multiple databases and/or wildcard (*) character(s).

Options: -wa Write the original entry in A for each overlap.

-wb Write the original entry in B for each overlap.

Useful for knowing what A overlaps. Restricted by -f and -r.

-loj Perform a "left outer join". That is, for each feature in A report each overlap with B. If no overlaps are found, report a NULL feature for B.

-wo Write the original A and B entries plus the number of base pairs of overlap between the two features.

Overlaps restricted by -f and -r. Only A features with overlap are reported.

-wao Write the original A and B entries plus the number of base pairs of overlap between the two features.

Overlapping features restricted by -f and -r. However, A features w/o overlap are also reported with a NULL B feature and overlap = 0.

-u Write the original A entry once if any overlaps found in B.

In other words, just report the fact >=1 hit was found.

Overlaps restricted by -f and -r.

-c For each entry in A, report the number of overlaps with B.

Reports 0 for A entries that have no overlap with B.

Overlaps restricted by -f and -r.

-v Only report those entries in A that have no overlaps with B.

Similar to "grep -v" (an homage).

-ubam Write uncompressed BAM output. Default writes compressed BAM.

-s Require same strandedness. That is, only report hits in B that overlap A on the same strand.

By default, overlaps are reported without respect to strand.

-S Require different strandedness. That is, only report hits in B that overlap A on the opposite strand.

By default, overlaps are reported without respect to strand.

-f Minimum overlap required as a fraction of A.

Default is 1E-9 (i.e., 1bp).

FLOAT (e.g. 0.50)

-F Minimum overlap required as a fraction of B.

Default is 1E-9 (i.e., 1bp).

FLOAT (e.g. 0.50)

-r Require that the fraction overlap be reciprocal for A AND B.

In other words, if -f is 0.90 and -r is used, this requires that B overlap 90% of A and A also overlaps 90% of B.

-e Require that the minimum fraction be satisfied for A OR B.

In other words, if -e is used with -f 0.90 and -F 0.10 this requires that either 90% of A is covered OR 10% of B is covered. Without -e, both fractions would have to be satisfied.

-split Treat "split" BAM or BED12 entries as distinct BED intervals.

-g Provide a genome file to enforce consistent chromosome sort order across input files. Only applies when used with -sorted option.

-nonamecheck For sorted data, don't throw an error if the file has different naming conventions for the same chromosome. ex. "chr1" vs "chr01".

-sorted Use the "chromsweep" algorithm for sorted (-k1,1 -k2,2n) input.

-names When using multiple databases, provide an alias for each that will appear instead of a fileId when also printing the DB record.

-filenames When using multiple databases, show each complete filename instead of a fileId when also printing the DB record.

-sortout When using multiple databases, sort the output DB hits for each record.

-bed If using BAM input, write output as BED.

-header Print the header from the A file prior to results.

-nobuf Disable buffered output. Using this option will cause each line of output to be printed as it is generated, rather than saved in a buffer. This will make printing large output files noticeably slower, but can be useful in conjunction with other software tools and scripts that need to process one line of bedtools output at a time.

-iobuf Specify amount of memory to use for input buffer. Takes an integer argument. Optional suffixes K/M/G supported. Note: currently has no effect with compressed files.

Notes: (1) When a BAM file is used for the A file, the alignment is retained if overlaps exist, and excluded if an overlap cannot be found. If multiple overlaps exist, they are not reported, as we are only testing for one or more overlaps.

ERROR: -b option given, but no database file specified. /var/spool/slurm/slurmd/job42896608/slurm_script: line 14: output_short_intron2_sorted.gff: command not found Troubleshooting is now completed!

The following files were created but are empty (zero bytes): test_bam_short_intron2 test_short_intron1

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hvicars commented 4 months ago

I reran the first command by submitting a slurm job sh file:

bedtools intersect -a 16hrAligned.out.sorted.bed -b output_short_intron1_sorted.gff -sorted -wa -u -f 1 > test_short_intron1

I reran the first command in the directory where I ran JUM_A.sh

This was the result: Tool: bedtools intersect (aka intersectBed) Version: v2.27.1 Summary: Report overlaps between two feature files.

Usage: bedtools intersect [OPTIONS] -a <bed/gff/vcf/bam> -b <bed/gff/vcf/bam>

Note: -b may be followed with multiple databases and/or 
wildcard (*) character(s).

Options: -wa Write the original entry in A for each overlap.

-wb Write the original entry in B for each overlap.
    - Useful for knowing _what_ A overlaps. Restricted by -f and -r.

-loj    Perform a "left outer join". That is, for each feature in A
    report each overlap with B.  If no overlaps are found, 
    report a NULL feature for B.

-wo Write the original A and B entries plus the number of base
    pairs of overlap between the two features.
    - Overlaps restricted by -f and -r.
      Only A features with overlap are reported.

-wao    Write the original A and B entries plus the number of base
    pairs of overlap between the two features.
    - Overlapping features restricted by -f and -r.
      However, A features w/o overlap are also reported
      with a NULL B feature and overlap = 0.

-u  Write the original A entry _once_ if _any_ overlaps found in B.
    - In other words, just report the fact >=1 hit was found.
    - Overlaps restricted by -f and -r.

-c  For each entry in A, report the number of overlaps with B.
    - Reports 0 for A entries that have no overlap with B.
    - Overlaps restricted by -f and -r.

-v  Only report those entries in A that have _no overlaps_ with B.
    - Similar to "grep -v" (an homage).

-ubam   Write uncompressed BAM output. Default writes compressed BAM.

-s  Require same strandedness.  That is, only report hits in B
    that overlap A on the _same_ strand.
    - By default, overlaps are reported without respect to strand.

-S  Require different strandedness.  That is, only report hits in B
    that overlap A on the _opposite_ strand.
    - By default, overlaps are reported without respect to strand.

-f  Minimum overlap required as a fraction of A.
    - Default is 1E-9 (i.e., 1bp).
    - FLOAT (e.g. 0.50)

-F  Minimum overlap required as a fraction of B.
    - Default is 1E-9 (i.e., 1bp).
    - FLOAT (e.g. 0.50)

-r  Require that the fraction overlap be reciprocal for A AND B.
    - In other words, if -f is 0.90 and -r is used, this requires
      that B overlap 90% of A and A _also_ overlaps 90% of B.

-e  Require that the minimum fraction be satisfied for A OR B.
    - In other words, if -e is used with -f 0.90 and -F 0.10 this requires
      that either 90% of A is covered OR 10% of  B is covered.
      Without -e, both fractions would have to be satisfied.

-split  Treat "split" BAM or BED12 entries as distinct BED intervals.

-g  Provide a genome file to enforce consistent chromosome sort order
    across input files. Only applies when used with -sorted option.

-nonamecheck    For sorted data, don't throw an error if the file has different naming conventions
        for the same chromosome. ex. "chr1" vs "chr01".

-sorted Use the "chromsweep" algorithm for sorted (-k1,1 -k2,2n) input.

-names  When using multiple databases, provide an alias for each that
    will appear instead of a fileId when also printing the DB record.

-filenames  When using multiple databases, show each complete filename
        instead of a fileId when also printing the DB record.

-sortout    When using multiple databases, sort the output DB hits
        for each record.

-bed    If using BAM input, write output as BED.

-header Print the header from the A file prior to results.

-nobuf  Disable buffered output. Using this option will cause each line
    of output to be printed as it is generated, rather than saved
    in a buffer. This will make printing large output files 
    noticeably slower, but can be useful in conjunction with
    other software tools and scripts that need to process one
    line of bedtools output at a time.

-iobuf  Specify amount of memory to use for input buffer.
    Takes an integer argument. Optional suffixes K/M/G supported.
    Note: currently has no effect with compressed files.

ERROR: -b option given, but no database file specified. /var/spool/slurm/slurmd/job42905970/slurm_script: line 12: output_short_intron1_sorted.gff: command not found

The file output_short_intron1_sorted.gff is in my directory and is 1.3 MB, but the test_short_intron1 file that was created is empty (zero bytes).

qqwang-berkeley commented 3 months ago

The command should be in one line: bedtools intersect -a 16hrAligned.out.sorted.bed -b output_short_intron1_sorted.gff -sorted -wa -u -f 1 > test_short_intron1

that is why you got that error "command not found"

On Fri, Apr 19, 2024 at 2:01 PM hvicars @.***> wrote:

I reran the first command by submitting a slurm job sh file:

bedtools intersect -a 16hrAligned.out.sorted.bed -b output_short_intron1_sorted.gff -sorted -wa -u -f 1 > test_short_intron1

I reran the first command in the directory where I ran JUM_A.sh

This was the result: Tool: bedtools intersect (aka intersectBed) Version: v2.27.1 Summary: Report overlaps between two feature files.

Usage: bedtools intersect [OPTIONS] -a <bed/gff/vcf/bam> -b <bed/gff/vcf/bam>

Note: -b may be followed with multiple databases and/or wildcard (*) character(s).

Options: -wa Write the original entry in A for each overlap.

-wb Write the original entry in B for each overlap.

Useful for knowing what A overlaps. Restricted by -f and -r.

-loj Perform a "left outer join". That is, for each feature in A report each overlap with B. If no overlaps are found, report a NULL feature for B.

-wo Write the original A and B entries plus the number of base pairs of overlap between the two features.

Overlaps restricted by -f and -r. Only A features with overlap are reported.

-wao Write the original A and B entries plus the number of base pairs of overlap between the two features.

Overlapping features restricted by -f and -r. However, A features w/o overlap are also reported with a NULL B feature and overlap = 0.

-u Write the original A entry once if any overlaps found in B.

In other words, just report the fact >=1 hit was found.

Overlaps restricted by -f and -r.

-c For each entry in A, report the number of overlaps with B.

Reports 0 for A entries that have no overlap with B.

Overlaps restricted by -f and -r.

-v Only report those entries in A that have no overlaps with B.

Similar to "grep -v" (an homage).

-ubam Write uncompressed BAM output. Default writes compressed BAM.

-s Require same strandedness. That is, only report hits in B that overlap A on the same strand.

By default, overlaps are reported without respect to strand.

-S Require different strandedness. That is, only report hits in B that overlap A on the opposite strand.

By default, overlaps are reported without respect to strand.

-f Minimum overlap required as a fraction of A.

Default is 1E-9 (i.e., 1bp).

FLOAT (e.g. 0.50)

-F Minimum overlap required as a fraction of B.

Default is 1E-9 (i.e., 1bp).

FLOAT (e.g. 0.50)

-r Require that the fraction overlap be reciprocal for A AND B.

In other words, if -f is 0.90 and -r is used, this requires that B overlap 90% of A and A also overlaps 90% of B.

-e Require that the minimum fraction be satisfied for A OR B.

In other words, if -e is used with -f 0.90 and -F 0.10 this requires that either 90% of A is covered OR 10% of B is covered. Without -e, both fractions would have to be satisfied.

-split Treat "split" BAM or BED12 entries as distinct BED intervals.

-g Provide a genome file to enforce consistent chromosome sort order across input files. Only applies when used with -sorted option.

-nonamecheck For sorted data, don't throw an error if the file has different naming conventions for the same chromosome. ex. "chr1" vs "chr01".

-sorted Use the "chromsweep" algorithm for sorted (-k1,1 -k2,2n) input.

-names When using multiple databases, provide an alias for each that will appear instead of a fileId when also printing the DB record.

-filenames When using multiple databases, show each complete filename instead of a fileId when also printing the DB record.

-sortout When using multiple databases, sort the output DB hits for each record.

-bed If using BAM input, write output as BED.

-header Print the header from the A file prior to results.

-nobuf Disable buffered output. Using this option will cause each line of output to be printed as it is generated, rather than saved in a buffer. This will make printing large output files noticeably slower, but can be useful in conjunction with other software tools and scripts that need to process one line of bedtools output at a time.

-iobuf Specify amount of memory to use for input buffer. Takes an integer argument. Optional suffixes K/M/G supported. Note: currently has no effect with compressed files.

Notes: (1) When a BAM file is used for the A file, the alignment is retained if overlaps exist, and excluded if an overlap cannot be found. If multiple overlaps exist, they are not reported, as we are only testing for one or more overlaps.

ERROR: -b option given, but no database file specified. /var/spool/slurm/slurmd/job42905970/slurm_script: line 12: output_short_intron1_sorted.gff: command not found

The file output_short_intron1_sorted.gff is in my directory and is 1.3 MB, but the test_short_intron1 file that was created is empty (zero bytes).

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hvicars commented 3 months ago

Oh, thanks! I corrected that mistake and reran it. I received this error:

ERROR: Sort order was unspecified, and file 16hrAligned.out.sorted.bed is not sorted lexicographically. Please rerun with the -g option for a genome file. See documentation for details.

However, the test_short_intron1 file now contains 577 KB.